Diversity Supplement to Structural Dynamics of Cardiac Myosin-Binding Protein C Regulation

项目名称：心肌肌球蛋白结合结构动力学的多样性补充 蛋白C的调节。 肌球蛋白结合蛋白C(MyBP-C)在心脏功能调节中发挥重要作用。 肥厚性心肌病中MyBP-C基因突变导致的磷酸化和收缩功能缺陷 心肌病(HCM)和心力衰竭时的磷酸化减少。我们的目标是了解分子 肌肉的生物物理学，特别强调心脏，并训练下一代肌肉 生物物理学家，包括不同的受训人员。家长研究项目和多样性补充要求 关于蛋白质相互作用的作用和结构动力学调节功能的基本问题 心肌。为了深入了解MyBP-C机制中涉及的结构与功能的相关性 在生理和病理背景下，我们将探索这些蛋白的肌动蛋白-肌球蛋白-MyBP-C复合体在 具有不同结合、磷酸化和HCM突变的溶液。我们的核心技术是站点定向的 光谱，应用于纯化的MyBP-C和肌动蛋白/肌球蛋白细丝。我们将应用创新互补 用定点标记和光谱学的方法将蛋白质结合、结构动力学和功能联系起来。 我们将检验中心假设，即N-末端MyBP-C的磷酸化和HCM突变改变 MyBP-C单独以及与肌动蛋白和肌球蛋白相互作用时具有显著的功能结构特性。相关 为了资助目标1，多样性补充的第一阶段侧重于使用光谱方法来 准确测量纯化的MyBP-C在发生磷酸化的M-结构域内的结构动力学， 主要是通过测量纳米距离和分子无序。主要的重点是检测 磷酸化、HCM突变和肌动蛋白引起的MyBP-C M结构域内构象(结构)变化 或肌球蛋白结合(功能)。通过将探针的位置包括在M-域中，候选人将测量 从我们的计算模拟中预测的结构性变化。将制备荧光标记的MyBP-C 用时间分辨的方法获得荧光寿命。在第二阶段，候选人将学习新的 光谱数据拟合分析的技巧，以确定N端的探针间距离和无序度 MyBP-C。第三阶段将通过提供结构的分子细节来对目标1和目标2做出贡献 添加肌钙蛋白、原肌球蛋白和肌球蛋白模拟生理学的肌动蛋白-MyBP-C复合体的动力学 心肌细丝在肌肉中的状态。应聘者将系统地建立模型系统 复杂性，从肌动蛋白结合到细丝结合的MyBP-C，具有低(舒张期)或高(收缩期)激活剂， 以及结合心肌肌球蛋白，提供在肌丝水平上的关键见解 了解肌肉细胞的基本机制。MyBP-C对Thin的调节作用的光谱研究 细丝将决定蛋白质的相互作用和结构动力学。这个项目是以基础为基础的 生物物理机制，但MyBP-C已成为心脏病的治疗靶点。因此，我们的工作 为使用我们独特的光谱方法开发药物治疗筛选奠定了基础。

Title of project: DIVERSITY SUPPLEMENT TO STRUCTURAL DYNAMICS OF CARDIAC MYOSIN-BINDING PROTEIN C REGULATION. Myosin-binding protein C (MyBP-C) plays a major role in the modulation of cardiac function by its phosphorylation and causes deficits in contractile function due to MyBP-C mutations in hypertrophic cardiomyopathy (HCM) and reduced phosphorylation in heart failure. Our goal is to understand the molecular biophysics of muscle, with particular emphasis on the heart, and to train the next generation of muscle biophysicists, inclusive of diverse trainees. The parent research project and diversity supplement ask fundamental questions about the role of protein interactions and structural dynamics that regulate function in cardiac muscle. To gain insight into the correlation of structure-function involved in MyBP-C mechanisms in physiological and pathological settings, we will probe the actin-myosin-MyBP-C complex of these proteins in solution with varied binding, phosphorylation, and HCM mutations. Our core technology is site-directed spectroscopy, applied to purified MyBP-C and actin/myosin filaments. We will apply innovative complementary methods in site-directed labeling and spectroscopy to correlate protein binding, structural dynamics and function. We will test the central hypothesis that phosphorylation and HCM mutations of N-terminal MyBP-C alter functionally significant structural properties of MyBP-C alone and as it interacts with actin and myosin. Related to parent grant Aim 1, the first period of the diversity supplement focuses on using spectroscopic approaches to accurately measure the structural dynamics within M-domain of purified MyBP-C, where phosphorylation occurs, primarily by measuring nanometer distances and molecular disorder. Major emphasis is placed on detection of conformational changes (structure) within MyBP-C’s M-domain due to phosphorylation, HCM mutation, and actin or myosin binding (function). By including the location of probes in M-domain, the Candidate will measure structural changes predicted from our computational simulations. Fluorescently-labeled MyBP-C will be prepared to acquire fluorescence lifetime using time-resolved methods. In the second period, the Candidate will learn new skills in spectroscopic data fitting analysis to determine probe-to-probe distances and disorder in N-terminal MyBP-C. The third period will contribute to both Aim 1 and Aim 2 by providing molecular details of the structural dynamics of the actin-MyBP-C complex with added troponin, tropomyosin, and myosin to mimic physiological conditions of the cardiac thin filament in muscle. The Candidate will systematically build in model system complexity, from actin-bound to thin filament-bound MyBP-C, with low (diastole) or high (systole) activator Ca2+, and upon binding of cardiac myosin, providing key insights at the myofilament level to be applied for understanding fundamental mechanisms in the muscle cell. Spectroscopic study of MyBP-C regulation of thin filaments will determine protein interactions and structural dynamics. This project is grounded in fundamental biophysics mechanisms, but MyBP-C has emerged as a therapeutic target for cardiac disease. Thus, of our work lays a foundation for development of screens for drug therapies using our unique spectroscopic approaches.